Cl- is the major anion in the aqueous humor. The final step in Cl- secretion is likely extrusion from the nonpigmented ciliary epithelial (NPE) cells through ion channels. These Cl- channels have been detected in the intact ciliary epithelium and in isolated NPE cells but only in low abundance, so that this site is likely a rate-limiting step to aqueous humor formation. Based on our volumetric, electrophysiologic and collaborative molecular biologic measurements conducted under hypotonic and isotonic conditions, we have formulated a hypothesis concerning ciliary epithelial Cl- secretion. We propose that the Cl channel CIC-3 and the Cl- channel regulator P1cln control the physiologic secretion of Cl-, that the same Cl- channels subserving aqueous humor formation are recruited during the response to hypotonic cell swelling, that these Cl- channels are regulated by protein kinase C, calcium/calmodulin and an epoxide, and that the regulation of these Cl- channels significantly modulates the rate of aqueous humor formation. We shall extend our studies of broken cell, isolated-cell and intact epithelial preparations, applying both electrophysiology and molecular biologic strategies. We shall study primary and/or continuous lines of NPB and pigmented ciliary epithelial (PE) cells (of human, rabbit, bovine, and cat origin) by patch clamping in the ruptured-membrane and perforated- membrane, whole cell modes and in the cell-attached and excised-patch modes. In addition, we shall monitor transport of the intact ciliary epithelium non-invasively with cell-attached patching. The objectives of the program are to test our hypothesis of ciliary epithelial Cl- secretion by addressing the following sets of issues (l) Do the NPB cells display only a single set of functional Cl-channels under hypotonic and isotonic conditions? (ii) Do the pigmented ciliary- epithelial (pE) cells display the same or different functional Cl- channels? Can transcripts and protein products for CIC-3 and pI-cln be detected in PE cells? What are the relative abundances of the functional Cl- channels and of these transcripts and protein products in the PE and NPE cells? (iii) What are the functional effects of overexpressing (by transfection) and of down-regulating (by antisense oligodeoxynucleotides and antibodies) the CIC-3 Cl- channel and the pI-cln Cl- channel regulator?